B-type natriuretic peptide (brain natriuretic peptide, BNP)belongs to the family of structurally similar, but genetically distinct natriuretic peptides (NPs) first described by de Bold et al. (Proc Soc Exp Biol Med 161:508-511 1979 and Life Science 28:89-94 1981). The NPs are protein hormones which possess potent diuretic, natriuretic and vasodilatory properties and have been reported as valuable diagnostic and prognostic markers in cardiovascular disease, particularly for patients in New York Heart Association (NYHA) classes I-IV congestive heart failure (CHF) (Boomsma et al. Cardiovascular Research 51:442-449 2001).
The human proBNP gene has been cloned by various groups (for example, Sudoh et al. Biochem Biophys Res Commun 159(3):1427-1434 1989). The protein products of the proBNP gene are major diagnostic markers of congestive heart failure. Expression of the BNP protein within the cell (cardiomyocyte) occurs as a 134 amino acid residue preproBNP. Cleavage of the 26 amino acid signal peptide from the N-terminal results in proBNP which is 108 amino acid residues in length. Prior to release into the blood, proBNP-undergoes a final protein processing step where a 32 amino acid C-terminal peptide (BNP-32) is cleaved. BNP-32 is the bioactive hormone. The remaining amino acid residues 1-76 is termed NT-BNP or NT-proBNP. NT-BNP also circulates in the plasma and like BNP is an important marker of ventricular dysfunction (Hunt et al. Biochem Biophys Res Commun 14:1175-1183 1995 and Hunt et al. Clin Endocrinology 47:287-296 1997).
Many studies have demonstrated the clinical utility of measuring plasma concentrations of NPs, including NT-proBNP. NPs have been suggested as the biomarkers of choice for diagnosis and risk stratification of patients with heart failure (Boomsma et al. Cardiovascular Research 51:442-449 2001; Hunt et al. Clin Endocrinology 47:287-296 1997; Clerico et al. Clinical Chemistry 46:1529-1534 2000; Mair et al. Clin Chem Lab Med 39:571-588 2001; Sagnella Ann Clin Biochem 38:83-93 2001; Selvais et al. Eur J Clin Invest 28:636-642 1998; McDonagh et al. Heart 86:21-26 2001). Several studies have shown the utility of using NP measurements to identify patients with left ventricular dysfunction, even amongst patients who are asymptomatic (NYHA class I) and it has been suggested that NP measurements as a screening tool may help effectively target patients within high risk heart failure groups (coronary artery disease, hypertension, diabetes, elderly) who will require follow-up assessment and treatment (Hunt et al. Clin Endocrinology 47:287-296 1997; Hughes et al. Clinical Science 96:373-380 1999; Omland et al. Heart 76:232-237 1996; McDonagh et al. Lancet 351:9-13 1998; Schulz et al. Scand J Clin Lab Invest 61:33-42 2001; Talwar et al. Eur Heart J 20:1736-1744 1999; Hystad et al. Acta Physiol Scand 171:395-403 2001; Hobbs et al. BMJ 324:1498 2002). NPs have been shown to have good prognostic value with regards to both morbidity and mortality in heart failure. Several studies have also demonstrated the utility of NP measurements in the prediction of left ventricular dysfunction and survival following acute myocardial infarction (Richards et al. Circulation 97:1921-1929 1998; Luchner et al. Hypertension 39:99-104 2002; Campbell et al. Intern Med J 31:211-219 2001; Nilsson et al. Am Heart J 143:696-702 2002). Monitoring NP levels may also provide guidance in tailoring therapies to meet the required intensity of the individual patient and in monitoring therapeutic efficacy (Richards et al. J Am Coll Cardiol 37:1781-1787 2001; Troughton et al. Lancet 355:1126-1130 2000).
The NT-proBNP assays disclosed in co-pending application Ser. Nos. 10/299,977 and 10/300,733 employ a sandwich ELISA technique to measure circulating NT-proBNP in human plasma. Such immunoassay techniques provide a quantitative estimate of concentration by direct comparison with a standard material. However, there are no pre-existing reference methods by which to calibrate standards for the majority of analytes determined by immunoassay. In the absence of such reference methods, calibration requires a source of purified analyte (see The Immunoassay Handbook, edited by David Wild, Stockton Press, 1994, especially pages 54-57 for a discussion of calibration and standardization).
In the NT-proBNP immunoassay the purified analyte required is NT-BNP. The production of recombinant NT-proBNP is not easily achieved based upon the short amino acid sequence.
Hunt et al. (Clinical Endocrinology 47:287-295 1997)teaches a method for identification of NT-proBNP in human plasma using a radioimmunoassay technique. A synthetic human peptide of amino acid residues of 1-21 of proBNP was used as a standard in Hunt et alls radioimmunoassay (see FIG. 1of Hunt et al.).
WO 00/45176 (Karl et al.) discloses a method for recombinant expression of NT-proBNP in bacteria (E. coli)using N-terminal histidine tags (see example 1 of Karl et al.). This purified N-terminal proBNP was expressed to be used as a calibrator in the immunoassay methods of Karl et al.
There are some disadvantages to the expression of recombinant proteins using tags. The presence of tags at the N-terminus can alter the three-dimensional conformation and thus alter the biological activity of the protein. Additionally, affinity chromatography is often the purification step when carrying out methods of expression using tagging. The chemicals used in affinity chromatography can also alter the protein conformation and thus the biological activity. It would be advantageous to avoid these problems by utilizing a method for expression of untagged recombinant proteins.
What is lacking in the art is a method to efficiently express high levels of a recombinant untagged NT-proBNP protein capable for use as a calibrator in NT-proBNP immunoassays.